PROJECT SUMMARY Emerging and re-emerging viruses cause a constant threat to global health. Discovery and characterization of cellular signaling pathways that regulate pathogenesis and host defense hold promise for revealing new strategies aimed at enhancing resistance to infection. There are no approved antiviral therapies available for coronaviruses, including SARS-CoV-2, that cause disease on a large scale, highlighting the need for innovative approaches to develop more broad-spectrum antivirals. Host-directed therapy (HDT) is an emerging approach in the field of anti-infectives. The strategy behind HDT is to interfere with host cell factors that are required by a pathogen for replication. Recent breakthroughs in somatic cell genetics have enabled genome-scale genetic knockout screens in human cells to identify cellular factors critical to infection and to dissect innate immune pathways. The pooled genetic knockout approach has several key advantages. First, by using a genome-scale CRISPR library and using pseudotyped virus for entry and a SARS-CoV-2 replicon for RNA replication and transcription, only those genes are selected whose knockout confers a strong resistance to virus infection. Second, because this approach relies on complete knockout of the gene of interest, we select only those genes that affect infection without being required for cellular viability and growth. In this competitive supplement, we propose to use these robust and unbiased knockout screening approaches to identify and thoroughly characterize novel host targets essential for infection by SARS-CoV-2. We expect that these genome-scale screens will elucidate promising cellular targets that could be used to develop host-directed antiviral therapy.